1. Field of the Invention
The present invention in the field of molecular biology and medicine relates to interfering RNA (RNAi) compositions and methods of using them to reduce the expression of the Met oncogene, a receptor for hepatocyte growth factor/scatter factor (HGF/SF), in tumor cells. This promotes apoptosis and results in inhibition of tumor cell growth, invasion and metastasis.
2. Description of the Background Art
Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic factor that induces a wide range of cellular functions, including proliferation, migration, angiogenesis and morphogenesis (Gherardi, E et al., Cancer Cells 3:227-232, 1991; Takebayashi, T et al., J Cell Biol 129:1411-1419, 1995; Jeffers, M et al., Cell Growth Differ 7:1805-1813, 1996; Sonnenberg, E et al., J Cell Biol 123:223-235, 1993) Met, the only known receptor for the HGF/SF ligand, can mediate signaling to downstream molecules (Birchmeier, C et al. Trends Cell Biol 8:404-410, 1998; Stewart, F Rev Reprod 1:144-148, 1996; Furge, K A et al., Oncogene 19:5582-5589, 2000; Birchmeier, C et al., Nat Rev Mol Cell Biol 4:915-925, 2003; Bottaro, D et al., Science 251:802-804, 1991).
HGF/SF is produced mainly by mesenchymal cells, while Met is preferentially expressed in epithelial and endothelial cells (Jeffers, M et al., Oncogene 13:853-856, 1996; Yang, X M et al., Lab Invest 73:483-491, 1995; Sonnenberg, E et al., Exs 65:381-394, 1993). In many types of tumor cells, Met signaling is activated through ligand-dependent autocrine or paracrine mechanisms (Park, W S et al., Apmis 108:195-200, 2000; Morello, S et seq., J Cell Physiol 189:285-290, 2001). Enhanced signal transduction via the stimulation of this receptor contributes to the malignant phenotype. Activating mutations in the Met receptor, first discovered in human papillary renal carcinomas (Schmidt, L et al., Nat Genet 16:68-73, 1997) have now been discovered in several different types of cancers and metastatic lesions. In mouse models, these mutations induce transformation, proliferation and invasion in vitro, as well as tumorigenicity and metastasis in vivo (Jeffers, M et al., Oncogene 17:2691-2700, 1998; Jeffers, M et al., Proc Natl Acad Sci USA 94:11445-11450, 1997)
HGF/SF binding to Met activates signaling downstream (Ponzetto, C et al., Cell 77:261-271, 1994) through various pathways such as the Ras mitogen-activated protein kinase (MAPK) pathways through Grb2-SOS complex formation (Ponzetto, C et al, J Biol Chem 271:14119-14123, 1996) or the Ras and Rac pathways (Ridley, A J et al., Mol Cell Biol 15:1110-1122, 1995) responsible for tubulo-morphogenesis (Sachs, M et al., J Cell Biol 133:1095-1107, 1996) and cell spreading/actin reorganization. Likewise, the STAT pathway, particularly STAT3 activation, is required for HGF/SF-Met-mediated growth in soft agar (Zhang, Y W et al., Oncogene 21:217-226, 2002), and PI3 kinase activation followed by Akt phosphorylation contributes to the prevention of apoptotic cell death (Xiao, G H et al., Proc Natl Acad Sci USA 98:247-252, 2001; Fan, S et al., Mol Cell Biol 21:4968-4984, 2001). In addition, HGF/SF-Met signaling can up-regulate the production of matrix metalloproteinases and urokinase that induce the degradation of extracellular matrices and basement membrane and enhance tumor invasion and metastasis (Harvey, P et al., Br J Cancer 83:1147-1153, 2000; Kermorgant, S et al., Carcinogenesis 22:1035-1042, 2001). Moreover, the activation of Met protein (Jeffers et al., 1997, supra) is involved in the induction of blood vessel formation in tumors by increasing the production of VEGF (Rosen, E M et al., Ciba Found Symp 212:215-226, 227-229, 1997; Tomita, N et al., Circulation 107:1411-1417, 2003) and by simultaneously shutting off the anti-angiogenesis factor thrombospondin-1 (Zhang, Y W et al., Proc Natl Acad Sci USA in press 2003; U.S. Ser. No. 60/484,676).
Since HGF/SF-Met signaling is implicated in a wide range of tumors and regulates biological activities that contribute to the tumor cell malignancy, targeting the Met receptor has become a subject of interest in the field of cancer biology (Birchmeier et al., supra). Cao et al. (Cao, B et al., Proc Natl Acad Sci USA 98:7443-7448, 2001) showed that HGF/SF-neutralizing antibodies efficiently block in vitro scattering and branching morphogenesis and can suppress the in vivo growth of HGF/SF-Met signal-dependent glioblastoma cell xenografts in nude mice. This suggests that interrupting the autocrine and/or paracrine HGF/SF-Met signaling in tumors that depend on this pathway is a potential intervention strategy.
The administration of c-met-antisense oligonucleotides is another molecular approach used to block Met function in cancer cells. A recent report showed that (a) c-met-antisense oligonucleotides decreased Met protein levels in the LoVo human colon cancer cell line and (b) apoptotic cell death (induced by serum deprivation) was more prominent in antisense-treated cells than in controls (Kitamura, S et al., Br J Cancer 83:668-673, 2000).
Production of the dominant negative (“DN”) forms of the Met protein is another approach to suppressing Met function. DN-Met in DA3 mouse mammary adenocarcinoma cells reduces tumorigenicity in vivo and metastatic potential (Firon, M et al., Oncogene 19:2386-2397, 2000). By using DN-Met that has inactivating mutations at both ATP binding sites in the kinase domain and at two important Tyr residues in the multidocking site, Furge et al. showed that inhibition of the Met receptor can suppress Ras-mediated metastasis (Furge, K A et al., Proc Natl Acad Sci USA 98:10722-10727, 2001). Ribozymes that target Met mRNA constitute a more direct approach to suppressing Met expression. Abounader et al. designed a hammerhead ribozyme against Met and showed that reduction of Met expression by the ribozyme suppressed anchorage-independent in vitro colony formation and in vivo tumorigenicity (Abounader, R et al., J Natl Cancer Inst 91:1548-1556, 1999. These investigators also showed that Met targeting by the ribozyme suppressed tumor growth and angiogenesis and, in turn, promoted apoptotic cell death (Abounader, R et al., Faseb J 16: 108-110, 2002). Christensen et al. disclosed that selective inhibition of Met tyrosine kinase activity using ATP-competitive small molecules had anti-tumor effects and thus anti-cancer therapeutic potential (Christensen, J G et al., Canc Res 63:7345-7355, 2003).
RNA interference (RNAi) is a recently reported phenomenon that has developed into a new approach for elucidating gene function. RNAi is a sequence-specific, post-transcriptional, gene-silencing mechanism that is effected through double-stranded RNA (dsRNA) molecules homologous to a sequence of the target gene (Elbashir, S M et al., Nature 411:494-498, 2001; Fire, A et al., Nature 391:806-811, 1998; Tuschl, T et al., Genes Dev 13:3191-3197, 1999). Fragments of the dsRNA called “small interfering” RNAs (siRNAs) can rapidly induce loss of function, and only a few molecules are required in a cell to produce the effect (Fire et al., supra) through hybrid formation between a homologous siRNA and mRNA (Lin, S L et al., Curr Cancer Drug Targets 1:241-247, 2001). A member of the RNase III family of nucleases named dicer has been identified as being involved in processing (Bernstein, E et al., Nature 409:363-366, 2001). DNA vector-mediated RNAi technology has made it possible to develop therapeutic applications for use in mammalian cells (Sui, G et al., Proc Natl Acad Sci USA 99:5515-5520, 2002; McCaffrey, A P et al., Nature 418:38-39, 2002; Lee, N S et al., Nat Biotechnol 20:500-505, 2002). There have been several reports of delivery by retroviral vectors for stable expression (Barton, G. M et al., Proc Natl Acad Sci USA 99:14943-14945, 2002; Paddison, P J et al., Cancer Cell 2:17-23, 2002; Rubinson, D A et al., Nat Genet 33:401-406, 2003; Tiscornia, G et al., Proc Natl Acad Sci USA 100:1844-1848, 2003) or adenoviral vectors for transient expression (Xia, H et al., Nat Biotechnol 20:1006-1010, 2002).
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.